Bottom Line:
A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase.PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites.The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl.

ABSTRACTThe semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

Figure 2: The Sp domain of ADAMTS-5 promotes PPS binding and an affinity increase(A) Schematic representation of ADAMTS-5 forms lacking sequential C-terminal domains. (B) Effect of PPS on the affinity between TIMP-3 and different forms of ADAMTS-5. TIMP-3 (0.5 nM) and ADAMTS-5 forms (0.5 nM, symbols as shown in A) were incubated with PPS (0.5–1000 nM for 1 h at 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (C) PPS (2.5 μM) was immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with various forms of ADAMTS-5 (0.4–25 nM, symbols as shown in A). Bound ADAMTS-5 was detected using an M2 anti-FLAG antibody.

Mentions:
The domains of ADAMTS-5 required for binding to PPS and for the affinity increase were examined using forms of the enzyme lacking sequential C-terminal non-catalytic domains (Figure 2A).

Figure 2: The Sp domain of ADAMTS-5 promotes PPS binding and an affinity increase(A) Schematic representation of ADAMTS-5 forms lacking sequential C-terminal domains. (B) Effect of PPS on the affinity between TIMP-3 and different forms of ADAMTS-5. TIMP-3 (0.5 nM) and ADAMTS-5 forms (0.5 nM, symbols as shown in A) were incubated with PPS (0.5–1000 nM for 1 h at 37°C) and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (C) PPS (2.5 μM) was immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with various forms of ADAMTS-5 (0.4–25 nM, symbols as shown in A). Bound ADAMTS-5 was detected using an M2 anti-FLAG antibody.

Mentions:
The domains of ADAMTS-5 required for binding to PPS and for the affinity increase were examined using forms of the enzyme lacking sequential C-terminal non-catalytic domains (Figure 2A).

Bottom Line:
A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase.PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites.The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl.

ABSTRACTThe semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.